The present invention generally relates to the imaging of the body portions of animals, and specifically to the field of phosphorimetry.
This invention is based on the quenching by molecular oxygen of the luminescence of various chemical compounds. This quenching effect can be used for imaging the distribution and concentration of oxygen in body portions of animals, including humans. Such information is indicative of tissue structure and anomalies, defects and diseases associated therewith. For example, certain disease states are characterized by the alteration of oxygen pressure in the involved tissue. The reader is referred to U.S. Pat. No. 4,947,850 for further discussion of the Background of the present invention.
xe2x80x9cLuminescencexe2x80x9d is the emission of light radiation from a species after that species has absorbed radiation. Luminescence involves the conversion of a molecule to an unstable, excited state. The emission of light arises on the return of the compound to its normal state. xe2x80x9cPhotoluminescencexe2x80x9d refers to luminescence which is associated with excitation by light of substantially short wavelengths. The light emitted from the excited species and which is confined to the period of excitation is fluorescence. The emitted light which persists after excitation has ceased is phosphorescence, or afterglow.
Phosphorescence of certain chemical compounds is quenched by oxygen according to the Stern-Volmer relationship which is stated as follows:
T0/T=1+kQ*T0PO2
where T0 and T are the phosphorescence lifetimes in the absence of oxygen, PO2 is the oxygen pressure for a lifetime of T, and kQ is the quenching constant. The constant kQ is related to the frequency of collisions between the excited triplet state molecule and molecular oxygen and the probability of energy transfer occurring when these molecules collide.
Various and often countervailing considerations are associated with the design, selection and/or preparation of materials for use as phosphorescent probes to study tissue oxygenation. It is generally required that the probe comprises a phosphorescent chromophore, and that the probes be soluble in aqueous solution, for example, physiological media.
The phosphorescent chromophore is the phosphorescent portion of the probe molecule. The chromophore can be converted to the triplet state (T1) by light absorption, followed by return to the ground state either with light emission (phosphorescence and/or delayed fluorescence) or by energy transfer to molecular oxygen.
Phosphorescent oxygen probes which are currently in use are generally based on Group VIII metals, e.g., palladium (Pd) and platinum (Pt) derivatives of porphyrins. See D. F. Wilson et al., J. Appl. Physiol., Vol. 70(6), pp. 2691-92 (1991). The Group VIII metalloporphyrins are advantageous in that they generally have high quantum yields which correspond to the fraction of excited molecules that phosphoresce. The Group VIII metalloporphyrins also possess desirable phosphorescence lifetimes and oxygen-quenching constants. However, these compounds possess serious drawbacks when considered for application to clinical measurements. In this connection, the absorption band of the Group VIII metal porphyrins is generally located at less than about 600 nanometers (nm). Other chromophores which occur naturally in living tissue, for example, hemoglobin, myoglobin and cytochrome, also have absorption bands at wavelengths less than about 600 nm. Due to the overlap in the wavelengths of the absorption bands, the naturally occurring chromophores absorb energy, for example, light, which is used to convert the Group VIII metalloporphyrins from the ground state to the triplet state. This prevents substantial excitation of the probe compounds.
Moreover, penetration of the excitation energy into the tissue is limited to about 50 to about 100 micrometers (xcexcm) when the excitation light is about 400 nm, and about 500 to about 1,000 xcexcm when the excitation light is about 560 nm. The penetration limitation is due, at least in part, to the tendency of chromophores which occur naturally in vivo, for example, hemoglobin, to absorb the excitation energy. The absorbance of the naturally-occurring chromophores generally decreases rapidly at wavelengths of greater than about 600 nm which is generally also the absorbance maxima of the currently used phosphorescing probe compounds.
The penetration limitation of excitation energy permits oxygen measurements of only the surface layer of tissue or substantially optically clear tissue, for example, eye tissue. The use of currently available phosphorescing compounds for imaging tissue oxygen is therefore generally limited to clinical pathologies of eye tissue and/or those lying right on or very near the surface of tissue.
Phosphorescent chromophores typically comprise a multiplicity of aromatic ring units. These aromatic ring units generally render the phosphorescent compounds substantially hydrophobic with little or no water solubility. However, it is generally required that phosphorescent compounds for imaging tissue oxygen be hydrophilic and soluble in aqueous solution, for example, physiological media. This aqueous solubility permits the compounds to circulate throughout the circulatory system of the host patient and be delivered to various tissue sites for subsequent excitation and examination and diagnosis of the involved tissue. The hydrophobicity of the currently available phosphorescing compounds generally limits their utility for clinical measurement of tissue oxygenation.
There is thus a need for phosphorescing compounds for studying tissue oxygenation which possess absorbance bands of greater than about 600 nm, this being the absorption maxima of prior art phosphorescing compounds. Moreover, there is a need for phosphorescing compounds for studying clinical pathologies at greater tissue depths and which are substantially soluble in aqueous solution, including physiological media.
In accordance with the above needs, the present invention provides improved methods and compounds for imaging internal body structures of animals, including humans. The methods and compounds of this invention provide numerous advantages over prior art methods and compounds. In a preferred embodiment, the present invention is directed to a compound for the measurement in vivo of oxygen in living tissue. The compound preferably comprises a chromophore which is capable of absorbing an amount of energy and subsequently releasing the energy as phosphorescent light. The chromophore preferably has an absorption band which is at a wavelength in the near infra-red window of living tissue, and the phosphorescence is quenched by molecular oxygen.
In a more preferred embodiment, the present invention is directed to a compound which is capable of phosphorescing and which has the formula 
where R1 is 2(3)-substituted aryl; R2 and R3 are independently hydrogen or are linked together to form substituted or unsubstituted aryl; and M is H2 or a metal.
In additional embodiments, the present invention is directed to a method for measuring the oxygenation of living tissue. The method comprises providing in vivo a phosphorescent compound having an energy absorption band at a wavelength in the near infra-red window of the tissue. The method further comprises causing said compound to phosphoresce and observing quenching by oxygen of the phosphorescence.
Preferred embodiments of methods and compounds taught and claimed herein provide significant clinical tools for examining, diagnosing and treating disease states which result in altered oxygen pressures in affected tissue. Compounds of the present invention are substantially hydrophilic in aqueous solution, for example, physiological media, and possess absorbance bands which are at a wavelength in the near infra-red window of living tissue. In view of their solubility and absorbance characteristics, the present methods and compounds overcome the drawbacks associated with prior art methods and compounds involving phosphorescing materials.
In still additional embodiments, the invention is directed to a method for preparing a compound of the formula
Porphxe2x80x94Xxe2x80x94Yxe2x80x94[xe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94]nxe2x80x94Hxe2x80x83xe2x80x83(II)
wherein Porph is a porphyrin selected from the group consisting of dihydroporphyrin and metalloporphyrin, X is a chemical bond or a linking group selected from the group consisting of xe2x80x94COxe2x80x94 and xe2x80x94NHCH2COxe2x80x94, Y is a chemical bond or xe2x80x94Oxe2x80x94, and n is an integer from about 8 to about 500, comprising:
(a) providing a compound of the formula
Porphxe2x80x94Xxe2x80x94Yxe2x80x94Zxe2x80x83xe2x80x83(III)
where Z is hydrogen, halo or hydroxy; and
(b) reacting the compound of formula III with PEG at a temperature and for a time to provide a PEG-substituted porphyrin.
Other features and advantages of the invention are described below in connection with the detailed description of preferred embodiments.
As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.
xe2x80x9cArylxe2x80x9d, xe2x80x9caryl groupxe2x80x9d and xe2x80x9caryl systemxe2x80x9d mean an unsaturated ring system characteristic of benzene. Preferred aryl groups or systems include ring systems of from about 6 to about 14 carbon atoms, and include phenyl, naphthyl, and anthryl, including phenanthryl.
xe2x80x9cAlkylxe2x80x9d means a saturated aliphatic hydrocarbon, either branched- or straight-chained. A xe2x80x9clower alkylxe2x80x9d is preferred having about 1 to about 6 carbon atoms. Examples of alkyl include methyl, ethyl, n-propyl, isopropyl, butyl, sec-butyl, t-butyl, amyl and hexyl.
xe2x80x9cAlkyloxyxe2x80x9d means an alkyl substituted oxy group. Lower alkyloxy groups are preferred.
xe2x80x9cSubstituted arylxe2x80x9d refers to an aryl group substituted with one or more substituent groups. In preferred embodiments, xe2x80x9csubstituent groupxe2x80x9d refers to chemical functional groups which are polar and which render the compounds substituted therewith hydrophilic and substantially soluble in aqueous media. These functional groups are also substantially reactive and are capable of being derivatized and/or of undergoing transformations using standard synthetic organic methodology. Non-limiting examples of preferred substituent groups include halo, carboxy (xe2x80x94CO2R, where R is hydrogen or alkyl), carboxamido, haloformyl (xe2x80x94C(xe2x95x90O)xe2x80x94X, where X is halo), hydroxy (xe2x80x94OH), amino (xe2x80x94NH2) and derivatives thereof, for example, mono- and dialkylamino, glycyl (including xe2x80x94NHCH2CO2H and xe2x80x94Cxe2x95x90O)CH2NH2), sulfonato (xe2x80x94SO2OR, where R is hydrogen or alkyl) and derivatives thereof, for example, halosulfonyl (xe2x80x94SO2X, where X is halo) and sulfonamide (xe2x80x94SO2NH2), and salts or derivatives thereof, including the reaction product of halosulfonyl and glycine. Preferred substituent groups are hydroxy and carboxy and derivatives and salts thereof.
In particularly preferred embodiments, xe2x80x9csubstituent groupxe2x80x9d refers to a hydrophilic ligand which is bonded to the chromophore through a covalent or coordinative bond and which renders the compounds hydrophilic and substantially soluble in aqueous media. Preferably, the ligand comprises sugar compounds or hydrophilic residues of flexible, polymeric compounds. The xe2x80x9cflexibilityxe2x80x9d of polymeric compounds refers to the torsional and/or rotational mobility of skeletal bonds in the chain portion of the polymer. In preferred embodiments, the chain portions of the polymeric compounds are highly flexible. Non-limiting examples of preferred flexible polymeric compounds include polymeric residues of proteins, for example, albumin, and polymers of substantially water-soluble monomers, for example, polymers or copolymers of ethylene glycol, propylene glycol, ethylene glycol amine and substituted or unsubstituted sugar compounds, including substituted or unsubstituted mono- and disaccharides. Amino-substituted sugars are preferred substituted sugar compounds, with glucosamine being particularly preferred.
In embodiments where the substituent groups comprise ligands which are covalently linked to the chromophore, the ligands are preferably linked to the chromophores through linking groups. Preferably, the linking groups comprise diradical functional groups, for example, sulfonyl (xe2x80x94SO2xe2x80x94) and carbonyl (xe2x80x94COxe2x80x94) diradicals.
xe2x80x9cHaloxe2x80x9d or xe2x80x9chalidexe2x80x9d means halogen. Preferred halogens include chloride, bromide and fluoride.
xe2x80x9cNear infra-red windowxe2x80x9d refers to the region in the light spectrum where light is only very slightly absorbed by tissue and is located between about 600 nm and about 1300 nm.
xe2x80x9cHost patientxe2x80x9d refers to animals, including humans, to which the compounds of the invention are administered for measuring tissue oxygen.
xe2x80x9cChromophorexe2x80x9d refers to a chemical group which, when present in an aromatic compound, imparts color to the compound by causing a displacement of, or appearance of, absorbent bands in the visible spectrum, and which is capable of being excited from a ground state to an excited state and returning to said ground state by either emitting phosphorescent light or by transferring energy to molecular oxygen.
When a term is used more than once in a chemical formula, each of its meanings is independent of the other.